Bone collagen matrix for zenogenic implants

ABSTRACT

Disclosed is a matrix material for implantation in a mammalian host comprising biocompatible mineral-free type I bone collagen, xenogenic to the host, and biodegradable therewithin. The matrix is manufactured from protein-extracted bone powder treated with certain swelling agents to increase its surface area and porosity. The matrix may be combined with osteogenic protein to induce reliably and reproducibly endochondral bone formation. It also can be used as a surface coat around implantable prosthetic devices to promote cellular ingrowth or as a carrier for sustained release of various therapeutic compositions.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending U.S. ApplicationSerial No. 315,342 pending filed Feb. 23, 1989 entitled OsteogenicDevices, the disclosure of which is hereby incorporated by referencewhich is a continuation-in-part of U.S. Ser. No. 232,630 pending filedAug. 15, 1988, which is a continuation-in-part of U.S. Ser. No. 179,406filed Apr. 8, 1988, allowed.

BACKGROUND OF THE INVENTION

This invention relates to a biocompatible, implantable material which isabsorbed naturally in vivo with minimal immunological reaction, and tothe methods for its production. More particularly, this inventionrelates to a superior collagenous bone matrix useful as a xenogenicimplant for use as an osteogenic device, as a bone particle coating forimplantable prostheses, as a delivery vehicle for the in vivo sustainedrelease of protein, and as a scaffold for anchorage-dependent cells.

A biocompatible, implantable material that can be resorbed in vivo couldbe used to promote conductive bone growth, induce osteogenesis whencombined with an osteoinductive protein, provide a substratum for invivo or in vitro growth of anchorage-dependent cells, or serve as acarrier for the sustained release of, for example, a therapeutic drug orantibiotic. Such a material must be biocompatible, that is, not inducean immunogenic/inflammatory response in vivo. Its physical structuremust allow cell infiltration, and it must have an in vivo resorptiontime appropriate for its function.

The potential utility of an osteogenic device capable of inducingendochondral bone formation in vivo has been recognized widely. It iscontemplated that the availability of such a device would revolutionizeorthopedic medicine, certain types of plastic surgery, and variousperiodontal and craniofacial reconstructive procedures.

The developmental cascade of bone differentiation in mammalian bonetissue is well documented in the art (Reddi, 1981, Collagen Rel. Res.1:209-226. Though the precise mechanisms underlying the phenotypictransformations are unclear, it has been shown that the naturalendochondral bone differentiation activity of bone matrix can bedissociatively extracted and reconstituted with inactive residualcollagenous matrix to restore full bone inducing activity (Sampath etal., 78 Proc. Natl. Acad. Sci. USA 7599-7603 (1981). Recently, theprotein factors hereafter referred to as osteogenic protein (OP)responsible for inducing osteogenesis have been purified, expressed inrecombinant host cells, and shown to be truly osteoinductive whenappropriately sorbed onto a matrix. (U.S. Patent Application No.179,406).

Studies have shown that while osteoinductive proteins are useful crossspecies, the demineralized bone matrix heretofore required for inducingendochondral bone formation is species specific (Sampath and Reddi(1983) PNAS 80:6591-6594). Implants of demineralized, extractedxenogenic bone matrix and OP invariably have resulted in a stronginflammatory response that has inhibited osteogenesis, presumably due toimmunogenic protein components in the bone matrix. Hence, successfulosteoinduction to date has required the use of allogenic bone matrix.This restriction on osteogenic devices is a serious limitation withrespect to human clinical use, as human bone is neither readilyavailable nor cost effective.

EPO 309,241 (published 3/29/89, filed 9/22/88, priority 9/25/87)discloses a device for inducing endochondral bone formation comprisingan osteogenic matrix extract, and a matrix carrier comprising 60-90%mineral component and 2-40% collagen.

U.S. Pat. No. 4,563,350, published Jan. 7, 1986, discloses a xenogenicosteogenic device comprising a bone-inducing extract that had beenPurified by gel filtration and ion exchange chromatography, and acollagenous matrix composed of approximately 90% trypsinized bovine bonematrix and 10% bovine dermal collagen. Endochondral bone formation issaid to require the presence of at least 10-15% dermal collagen in thedisclosed matrix.

Deatherage et al., (1987) Collagen Rel. Res. 7:2225-2231, purport todisclose an apparently xenogenic implantable device comprising a bovinebone matrix extract that has been minimally purified by a one-step ionexchange column and highly purified human Type-I placental collagen.

The current state of the art of materials used in surgical proceduresrequiring conductive bone repair, such as the recontouring or filling inof osseous defects, is disclosed by Deatherage (1988) J. OralMaxillofac. Surg. 17:395-359. All of the known implant materialsdescribed (hydroxyapatite, freeze-dried bone, or autogenous bone grafts)have little or no osteoinductive properties. Clearly, the ability toinduce osteogenesis is preferred over bone conduction for mostprocedures. Even when bone conduction is the indicated procedure forbone repair, a matrix consisting of non-immunogenic, extracted,xenogenic bone collagen heretofore has not been developed.

U.S. Pat. No. 4,795,467 discloses a bone repair composition comprisingcalcium phosphate minerals (preferable particle size of 100-2,000 μ) andatelopeptide, reconstituted, crosslinked fibrillocollagen. It purportsto be a non-antigenic, biocompatible, composition capable of fillingbony defects and promoting bone growth xenogenically.

U.S. Pat. No. 4,789,663 discloses a method of effecting conductive bonerepair comprising exposing the defect to fresh bone, and using xenogeniccollagen from bone and/or skin, wherein the collagen is enzymaticallytreated to remove telopeptides, and is artificially crosslinked.

The need to provide a "biological anchor" for implanted prostheses,particularly metallic implants, is well documented in the art. The stateof the art of prosthetic implants, disclosed by Specter (1987) J.Arthroplasty 2:163-177, generally utilizes porous coated devices, asthese coats have been shown to promote cellular ingrowth significantly.

EPO 169,001 (published 1/22/86, priority 07/17/84) claims acollagen-coated prosthesis wherein the coat comprises a purified,sterile, non-immunogenic xenogenic collagen preparation from bone orskin. Crosslinking is generally induced to reduce immunogenicity, oroccurs as a result of sterilization procedures.

U.S. Pat. No. 4,812,120 discloses a prosthetic device comprising a metalcore over which are applied successive polymers layers. The outer layercomprises a biopolymer having protruding collagen fibrils. Theprotruding fibrils are subject to damage upon implantation of thedevice.

Efficient in vitro growth of mammalian cells is often limited by thematerials used as the substration, substratum, or "scaffold" foranchorage-dependent cells. An effective matrix must be physiologicallyacceptable to the anchorage dependent cells, and it must also provide alarge available surface area to which the cells can attach.

GB Patent No. 2,178,447, published 02/11/87, claims a fibrous or porousfoam matrix comprising open or closed form fibers, with a pore size onthe order of 10-100 μ (matrix height is 50-500 μ). The fiber network isgenerated as a sheet which must then be modified if different scaffoldshapes are desired.

Strand et al. (Biotechnology and Bioengineering, V. 26, 503, 1984)disclose microcarrier beads for use as a matrix for anchorage dependentcells in a matrix perfusion cell culture. Bead materials tested wereDEAE or polyacrylamide. Surface area available was 250-300 cm^(2/) g andrequired a cell innoculaton of 10⁶ cells/ml.

U.S. Pat. No. 4,725,671 claims collagen fiber membranes suitable forcell culture, comprising soluble atelopeptide collagen fibers that aredried and preferably cross-linked.

The art has sought biocompatible sustained release vehicles with known,reliable "release" rates. Effective carriers must be biocompatible,water-insoluble, capable of trapping or otherwise holding thetherapeutic agent of interest, and have a resorption time in vivo thatmimics the desired release rate of the agent. Collagen and gelatin areattractive carriers for clinical use, primarily because of theirbiocompatible and biodegradable properties. (See, for example, EPO170,979, published 02/12/86, priority 8/07/84; and EPO 069,260,published 01/12/83, priority 6/25/81.) However, these biopolymers haveundesirable crosslinking properties that can make efficient synthesis ofappropriate carrier matrices difficult. (EPO 230,647 published 08/05/87,priority 12/27/85.)

It is an object of this invention to provide a biocompatible, in vivobiodegradable bone matrix, implantable in a mammalian host with nosignificant inhibitory immunogenic response. Another object is toprovide a biocompatible, in vivo biodegradable matrix capable ofcombining with an osteoinductive protein to produce endochondral boneformation in mammals, including humans. Still other objects are toprovide a superior material for coating implantable prothetic devices,to increase the cellular ingrowth into such devices, to provide abiocompatible, in vivo biodegradable matrix for use as a carrier ofsustained-release pharmaceutical compositions, wherein the resorptionrate of the matrix can be adjusted to match that of the pharmaceuticalagent, and to provide a biocompatible, in vivo biodegradable matrixcapable of acting as a scaffold or substratum for anchorage-dependentcells, wherein the surface area available for cell attachment can beadjusted. Yet another object of the invention is to provide a method forthe production of such matrix material.

These and other objects and features of the invention will be apparentfrom the description, drawings, and claims that follow.

SUMMARY OF THE INVENTION

This invention involves a matrix for implantation in a mammalian hostcomprising biocompatible, mineral-free, insoluble Type-I bone collagen,xenogenic to the host, which, when implanted in the host, isbiodegradable. As disclosed herein, the matrix may be combined withosteogenic protein to induce reliably and reproducibly endochondral boneformation in a mammalian body. It may also be used as a surface coataround implantable prosthetic devices to promote cellular ingrowth. Itcan act as a carrier for the sustained release of various compositionsin the mammalian body, and can provide a biocompatible substrate foranchorage-dependent cells.

The development of this matrix material resulted from the discovery ofkey features required for successful implantation of xenogenic bonematrix and osteogenic protein. Mammalian bone tissue growth requires theinflux, proliferation, and differentiation of migratory progenitor cellsat the site of the implant. Previous studies indicated that osteogenicdevices comprising substantially pure osteogenic protein anddemineralized, guanidine-extracted bone matrices must be particulate,with intraparticle interstices dimensioned to permit the influx,proliferation and differentiation of migratory cells. It is also knownthat osteogenic devices comprising xenogenic bone matrices induce littleor no endochondral bone formation in vivo. The inhibitory action ofxenogenic matrices was thought to be due to an immunogenic response toprotein components still present in the matrix (either the collagentelopeptides or associated non-collagenous glycoproteins.)

It has now been discovered that the overall intraparticle specificsurface area (surface area/unit mass) of the matrix itself is alsosignificant for xenogenic implants, even for allogenic implants ofcertain species. For example, allogenic, subcutaneous implants ofdemineralized, guanidine-extracted monkey bone matrix with OP isreported not to induce bone formation in the monkey. (Asperberg P., etal (1988) J. Bone Joint Surg. (br) 70-B, 625-627) .

Panels A and B of FIGS. 1 and 2 are scanning electron micrographsshowing the particle structure of demineralized, guanidine-extractedbone matrix from rat and calf, respectively. As can be seen from theSEMs, there is a significantly greater inherent porosity, orintraparticle surface area, in rat bone matrix over that of bovine bonematrix. It has been discovered that increasing porosity helps entrapprotein, and increases in intraparticle surface area can promoteosteogenic induction.

Thus, in one aspect, this invention comprises a matrix for implantationin a mammalian host comprising packed particles comprisingbiodegradable, biocompatible mineral-free, insoluble Type-I boneCollagen, xenogenic to the host, the particles being depleted innon-collagenous proteins, having a mean diameter within the range of 70μm-850 μm, and having an increased intraparticle surface area relativeto untreated material.

Another aspect of this invention involves methods of treatingdemineralized, guanidine extracted matrix particles with a swellingagent so as to increase the intraparticle porosity of the matrix toachieve the desired increase in matrix surface area. The matrixtreatments herein described increase the porosity of the matrixparticles, thereby altering the integrity of the particles. Thisalteration has the effect of potentially increasing the resorption timeof the matrix in vivo. Thus, one can alter treatment times to vary thematrix resorption rate in vivo (longer treatment times yield fasterresorption rates). The swelling agents include acids, for example,trifluoroacetic acid (TFA) and hydrogen fluoride (HF), and organicsolvents, for example, dichloromethane (DCM), acetonitrile (ACN) andisopropanol (IP). All these agents are known to have proteindenaturation properties and to swell insoluble proteins. Among these, apreferred swelling agent is DCM. The currently most preferred agent isDCM mixed with a small amount, e.g., 0.1%, of TFA.

Another preferred swelling agent is hydrogen fluoride, which is a knowndeglycosylating agent. Thus, in another aspect the invention provides amatrix for implantation in a mammalian host, comprising deglycosylatedType-I mineral-free bone collagen, xenogenic to the host, andbiodegradable and biocompatible in the host. This matrix preferably isin packed particle form. The particles preferably have a mean diameterof 70-850 μm, more preferable 150 μm-420 μm, and have an increasedintraparticle surface area relative to untreated material.

Treatment of the matrix with a swelling agent should be followed by anappropriate wash. Xenogenic bone matrices that have been treated with aswelling agent but left unwashed are less osteoinductive when implantedwith osteogenic protein in a mammalian host. Panels D and E in FIGS. 1and 2 show the dramatic effect the wash step has on intraparticlesurface area. Currently preferred washes include urea-containing bufferand water, or alternatively, a saline buffer.

Mammalian bone tissue growth requires the influx, proliferation anddifferentiation of migratory progenitor cells. Accordingly, in oneaspect, the invention comprises packed matrix particles, which may bedeglycosylated, defining interstices dimensioned to permit the influx,proliferation and differentiation of migratory cells, preferably havinga particle diameter that is in the range of 150-420 μm. In anotherpreferred aspect of the invention, the matrix comprises dispersedprotein, e.g., osteogenic protein, and is capable of inducingendochondral bone formation when implanted in a mammalian host.Preferred means of adsorbing the substantially pure osteogenic proteinonto the matrix particles include precipitation in cold ethanol fromguanidine HCl solution, or incubation in an acetonitrile/trifluoroaceticacid solution or in PBS, followed by lyophilization. The matrix may beshaped to span a non-union fracture or to fill in a defect in bone of amammalian host.

The biocompatible and in vivo biodegradable nature of the matrix alsomake it suitable for use as a delivery vehicle for the in vivo sustainedrelease of therapeutic drugs. The increased porosity can increase thematrix's ability to trap and absorb therapeutics. Moreover, it has beendiscovered that varying the swelling agent treatment times can alter theresorption rate of the matrix in vivo. Thus, this invention provides aneasily generated carrier source material of great versatility. In viewof this disclosure, those skilled in the art easily can create a carriermatrix having a specific, desired, reliable resorption time. They canthen adsorb the agent of interest onto the matrix using one of themethods disclosed herein, or any of the techniques known in the art, toprovide a sustained release vehicle with improved reliability in releaseof the therapeutic compound.

The particulate and porous nature of the material of this invention,along with its biocompatibility in mammalian hosts, permit its use atthe interface of an implanted prosthetic device and the surroundingmammalian tissue to promote cellular ingrowth. Moreover, the matrixstructure lends itself to increased durability during implantation ascompared with collagen fibrils commonly used in such compositions. Inview of this disclosure, those skilled in the art easily can create asurface coat for prosthetic devices having a specific, predeterminedporosity and increased durability. They can then attach the coat to theprosthetic core using any of the techniques known in the art. See, forexample, Cook et al., Clin. Ortho. Rel. Res. No. 232, p. 225, 1988. Thematrix further can comprise osteogenic protein if endochondral boneinduction is desired.

The nature of the matrix of this invention also makes it a superiorsubstratum for in vitro growth of anchorage-dependent cells. The matrixitself provides a physiologically acceptable surface for cellattachment, and the particle interstices and intraparticle porosityprovide significant increases in the surface area available for cellattachment over other known matrices. Moreover, the structure of thematrix of this invention allows one to vary the particle porosity asdesired. The cascade of pores present in this matrix promotes efficientnutrient access to cells, and increases the surface area available forcell attachment, thereby lowering the cell innoculant concentrationrequired in a cell perfusion system (See GB 2,178,447). In view of thisdisclosure, one skilled in the art efficiently can create abiocompatible matrix of choice, having a specific, known, desiredporosity.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of this invention, the various featuresthereof, as well as the invention itself, may be more fully understoodfrom the following description, when read together with accompanyingdrawings, in which:

FIGS. 1A through 1E are, respectively, scanning electron micrographs(350×) of: (1A) demineralized, guanidine-extracted rat bone matrix; (1B)demineralized, guanidine-extracted bovine bone matrix; 1C)demineralized, guanidine-extracted bovine bone matrix, further treatedwith hydrogen fluoride (HF) and washed; (1D) demineralized,guanidine-extracted bovine bone matrix, further treated withdichloromethane (DCM), and washed; (1E) demineralized,guanidine-extracted bovine bone matrix, further treated withdichloromethane (DCM), but unwashed; and (1F) demineralized,guanidine-extracted monkey bone matrix, further treated with hydrogenfluoride, and washed.

FIGS. 2A through 2E are, respectively, SEMs (5000×) of: (2A)demineralized, guanidine-extracted rat bone matrix; (2B) demineralized,guanidine-extracted bovine bone matrix; (2C) demineralized,guanidine-extracted bovine bone matrix, further treated with hydrogenfluoride, and washed; (2D) demineralized, guanidine-extracted bovinebone matrix, further treated with dichloromethane, and washed; and (2E)demineralized, guanidine-extracted bovine bone matrix, further treatedwith dichloromethane, but unwashed.

FIG. 3 is a bar graph of alkaline phosphatase activity (units/mgprotein) as a measure of osteogenesis in the presence of untreated andHF-treated bovine bone matrix; and

FIG. 4 is a bar graph of alkaline phosphatase activity as a measure ofosteogenesis in the presence of variously treated bovine matrixmaterials using DCM and DCM/TFA, and differing amounts of osteogenicprotein.

DETAILED DESCRIPTION

Practice of the invention requires the availability of bone, preferablymammalian bone, e.g., bovine. The bone is cleaned, demineralized,reduced to particles of an appropriate size, extracted to remove solubleproteins, sterilized, and otherwise treated as disclosed herein toproduce an implantable material useful in a variety of clinicalsettings.

Matrices of various shapes fabricated from the material of the inventionmay be implanted surgically for various purposes. Chief among these isto serve as a matrix for bone formation in various orthopedic,periodontal, and reconstructive procedures, as a sustained releasecarrier, or as a collagenous coating for implants. The matrix may beshaped as desired in anticipation of surgery or shaped by the physicianor technician during surgery. Thus, the material may be used forsubcutaneous, intraperitoneal, or intramuscular implants; it may beshaped to span a non-union fracture or to fill a bone defect. In boneformation or conduction procedures, the material is slowly absorbed bythe body and is replaced by bone in the shape of or very nearly theshape of the implant.

Various growth factors, hormones, enzymes, therapeutic compositions,antibiotics, and other body treating agents may be sorbed onto thecarrier material and will be released over time when implanted as thematrix material is slowly absorbed. Thus, various known growth factorssuch as EGF, PDGF, IGF, FGF, TGF alpha, and TGF beta may be released invivo. The material can be used to release antibiotics, chemotherapeuticagents, insulin, enzymes, or enzyme inhibitors.

Details of how to make and how to use the materials of the invention aredisclosed below.

A. Preparation of Demineralized Bone

Demineralized bovine bone matrix is prepared by previously publishedprocedures (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA80:6591-6595). Bovine diaphyseal bones (age 1-10 days) are obtained froma local slaughterhouse and used fresh. The bones are stripped of muscleand fat, cleaned of periosteum, demarrowed by pressure with cold water,dipped in cold absolute ethanol, and stored at -20° C. They are thendried and fragmented by crushing and pulverized in a large mill. Care istaken to prevent heating by using liquid nitrogen. The pulverized boneis milled to a particle size in the range of 70-850 μm, preferably 150μm-420 μm, and is defatted by two washes of approximately two hoursduration with three volumes of chloroform and methanol (3:1). Theparticulate bone is then washed with one volume of absolute ethanol anddried over one volume of anhydrous ether yielding defatted bone powder.The defatted bone powder is then demineralized by four successivetreatments with 10 volumes of 0.5 N HCl at 4° C for 40 min. Finally,neutralizing washes are done on the demineralized bone powder with alarge volume of water.

B. Guanidine Extraction

Demineralized bone matrix thus prepared is extracted with 5 volumes of 4M guanidine-HCl, 50mM Tris-HCl, pH 7.0 for 16 hr. at 4° C. Thesuspension is filtered. The insoluble material is collected and used tofabricate the matrix. The material is mostly collagenous in nature. Itis devoid of osteogenic or chondrogenic activity.

C. Xenogenic-Specific Treatments C1. Hydrogen Fluoride

The major component of all bone matrices is Type-I collagen. In additionto collagen, demineralized bone extracted as disclosed above includesnoncollagenous proteins which may account for 5% of its mass. Manynoncollagenous components of bone matrix are glycoproteins. In axenogenic matrix, these glycoproteins may present themselves as potentantigens by virtue of their carbohydrate content and may constituteimmunogenic and/or inhibitory components. A collagenous bone matrix maybe used for xenogenic implants if one first treats the immunogenic andinhibitorY components from the matrix with HF. Hydrogen fluoride is aknown deglycosylating agent, and as a strong acid and swelling agent,also alters intraparticle surface structure.

Bovine bone residue prepared as described above is sieved, and particlesof the appropriate size are collected. The samPle is dried in vacuo overP₂ O₅, transferred to the reaction vessel, and exposed to anhydroushydrogen fluoride (10-20 ml/g of matrix) by distillation onto the sampleat -70° C. The vessel is allowed to warm to 0° C and the reactionmixture is stirred at this temperature for 120 min. After evaporation ofthe HF in vacuo, the residue is dried thoroughly in vacuo over KOHpellets to remove any remaining traces of acid. Extent ofdeglycosylation can be determined from carbohydrate analysis of matrixsamples taken before and after treatment with HF, after washing thesamples appropriately to remove non-covalently bound carbohydrates. SDSextracted protein from HF treated material is negative for carbohydrateas determined by Con A blotting.

The deglycosylated bone matrix is next treated as set forth below:

(1) suspend in TBS (Tris-buffered saline) lg/200 ml, and stir at 4° Cfor 2 hrs; or in 6 M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0 (UTBS)and stir at room temperature (RT) for 30 min;

(2) centrifuge and wash with TBS or UTBS; and

(3) centrifuge; discard supernatant; water wash residue; and thenlyophilize.

C2. Trifluoroacetic acid

Like hydrogen fluoride, trifluoroacetic acid (TFA) is known to causeswelling of proteins. However, it does not effect deglycosylation.

Bovine bone residue, prepared as described above is sieved, andparticles of the appropriate size are extracted with various percentage(1.0% to 100%) of trifluoroacetic acid in water (v/v) at 0° C. or RT for1-2 hours with constant stirring. The treated matrix is filtered,lyophilized or washed with water/salt and then lyophilized.

C3. Dichloromethane

Dichloromethane (DCM) is an organic solvent capable of denaturingproteins without affecting their primary structure. It is a commonreagent in automated peptide synthesis, and is used in washing steps toremove unwanted components. DCM does not cause deglycosylation.

Bovine bone residue particles of the appropriate size prepared asdescribed above are incubated for one or two hours at 0° C, and also atRT for the same duration. After the treatment, the matrix is washed withthe standard 6M urea containing buffer, or water alone. Alternatively,the matrix is treated with DCM many times (X3) with short washes (20min. each) with no incubation.

C4. Acetonitrile

Acetonitrile (ACN) is an organic solvent, capable of denaturing proteinswithout affecting their primary structure. It is a common reagent inhigh performance liquid chromatography, and is used to elute proteinsfrom silica based columns by perturbing hydrophobic interactions.Acetonitrile does not cause deglycosylation.

Bovine bone residue particles of the appropriate size prepared asdescribed above are treated with 100% acetonitrile (1.0g/30ml) at roomtemperature for one to two hours with constant stirring. The treatedmatrix is then water washed, or washed with urea buffer, or 4M NaCl, andlyophilized.

C5. Isopropanol

Isopropanol also is an organic solvent capable of denaturing proteinswithout affecting their primary structure. It is a common reagent usedto elute proteins from silica HPLC columns. Isopropanol does not causedeglycosylation.

Bovine bone residue particles of the appropriate size prepared asdescribed above are treated with 100% isopropanol (1.0g/30ml) at roomtemperature for one to two hours with constant stirring. The treatedmatrix is then water washed, or washed with urea buffer or 4M NaClbefore being lyophilized.

C6. Combinations of Reagent

Separate bovine bone particle samples are treated with dichloromethane,or acetonitrile, and isopropanol, each of which contained 0.1%trifluoroacetic acid. The optimal conditions for the treatment areincubation with solvent/acid mixture at 0° C. or RT for one to two hourswith constant stirring. The treated matrix is then lyophilized withoutwash. Alternately, the treated matrices are washed with water or 4M saltbefore lyophilization. FIG. 4 illustrates the effectiveness of thesevarious treatments in converting bovine matrix to a material useful as abone formation matrix in rat. Further particulars of the evaluationprocedures are set forth below.

Treatment as set forth above in the swelling agents and other reagentsis effective to assure that the material is free of pathogens prior toimplantation.

The material is a fine powder, insoluble in water, comprisingnonadherent particles. It may be used simply by packing into the volumewhere new bone growth is desired, held in place by surrounding tissue.Then, immobilizing the region is sufficient to permit osteogenesis.Alternatively, the powder may be encapsulated in, e.g., a gelatin orpolylactic acid coating, which is adsorbed readily by the body. Thepowder may be shaped to a volume of given dimensions and held in thatshape by interadhering the particles using, for example, soluble,species biocompatible collagen.

II. IN VIVO RAT BIOASSAY

The functioning of the various xenogenic matrices can be evaluated withan in vivo rat bioassay. Studies in rats show the osteogenic effect inan appropriate matrix to be dependent on the dose of osteogenic proteindispersed in the matrix. No activity is observed if the matrix isimplanted alone. Demineralized, guanidine extracted xenogenic bonematrix materials of the type described in the literature are ineffectiveas a carrier, fail to induce bone, and produce an inflammatory andimmunological response when implanted unless treated as disclosed above.The following sets forth various procedures for preparing osteogenicdevices from control and matrix materials prepared as set forth above,and for evaluating their xenogenic utility.

A. Fabrication of Osteogenic Device

The osteogenic protein may be obtained using the methods disclosed inU.S. Patent Application No. 179,406 filed Apr. 8, 1988; PCT applicationNo. US89/01469 (entitled Biosynthetic Osteogenic Proteins and OsteogenicDevices Containing Them), and PCT Application No. US89/01453, (entitledOsteogenic Devices). Both PCT applications were filed Apr. 7, 1989.Alternatively, extracts rich in osteogenic protein useful in fabricatingdevices may be obtained as disclosed in U.S. Pat. No. 4,294,753 toUrist. The disclosure of these documents is incorporated herein byreference.

A1. Ethanol Precipitation

Matrix is added to osteogenic protein dissolved in guanidine-HCl.Samples are vortexed and incubated at a low temperature. Samples arethen further vortexed. Cold absolute ethanol is added to the mixturewhich is then stirred and incubated. After centrifugation (microfuge,high speed) the supernatant is discarded. The matrix is washed with coldconcentrated ethanol in water and then lyophilized.

A2. Acetonitrile Trifluoroacetic Acid Lyophilization

In this procedure, osteogenic protein in an acetonitrile trifluroaceticacid (ACN/TFA) solution was added to the carrier material. Samples werevigorously vortexed many times and then lyophilized. Osteogenic proteinwas added in varying concentrations, and at several levels of purity.This method is currently preferred.

A3. Urea Lyophilization

For those osteogenic proteins that are prepared in urea buffer, theprotein is mixed with the matrix material, vortexed many times, and thenlyophilized. The lyophilized material may be used "as is" for implants.

These procedures also can be used to adsorb other active therapeuticdrugs, hormones, and various bioactive species for sustained releasepurposes.

B. Implantation

The bioassay for bone induction as described by Sampath and Reddi (Proc.Natl. Acad. Sci. USA (1983) 80:6591-6595), herein incorporated byreference, may be used to monitor endochondral bone differentiationactivity. This assay consists of implanting the bovine test samplesxenogenically in subcutaneous sites in recipient rats under etheranesthesia. Male Long-Evans rats, aged 28-32 days, were used. A verticalincision (1 cm) is made under sterile conditions in the skin over thethoraic region, and a pocket is prepared by blunt dissection.Approximately 25 mg of the test sample is implanted deep into the pocketand the incision is closed with a metallic skin clip. The day ofimplantation is designated as day of the experiment. Implants wereremoved on day 12. The heterotropic site allows for the study of boneinduction without the possible ambiguities resulting from the use oforthotopic sites.

C. Cellular Events

Sucessful implants exhibit a controlled progression through the stagesof matrix induced endochondral bone development including: (1) transientinfiltration by polymorphonuclear leukocytes on day one; (2) mesenchymalcell migration and proliferation on days two and three; (3) chondrocyteappearance on days five and six; (4) cartilage matrix formation on dayseven; (5) cartiliage calcification on day eight; (6) vascular invasion,appearance of osteoblasts, and formation of new bone on days nine andten; (7) appearance of osteoblastic and bone remodeling and dissolutionof the implanted matrix on days twelve to eighteen; and (8)hematopoietic bone marrow differentiation in the ossicle on daytwenty-one. The results show that the shape of the new bone conforms tothe shape of the implanted matrix.

D. Histological Evaluation

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Implants are fixed in BouinsSolution, embedded in paraffin, and cut into 6-8 μm sections. Stainingwith toluidine blue or hemotoxylin/eosin demonstrates clearly theultimate development of endochondral bone. Twelve day implants areusually sufficient to determine whether the implants contain newlyinduced bone.

E. Biological Markers

Alkaline phosphatase activity may be used as a marker for osteogenesis.The enzyme activity may be determined spectrophotometrically afterhomogenization of the implant. The activity peaks at 9-10 days in vivoand thereafter slowly declines. Implants showing no bone development byhistology have little or no alkaline phosphatase activity under theseassay conditions. The assay is useful for quantitation and obtaining anestimate of bone formation quickly after the implants are removed fromthe rat. Alternatively, the amount of bone formation can be determinedby measuring the calcium content of the implant.

Results

The histological evaluation of implants made using HF- or DCM-treatedbone matrices is given in Table 1 and in FIG. 3. The osteogenic protein(OP) used in these experiments was isolated by the method disclosed inU.S. Patent Application No. 179,406. Experiments were performed usingeither moderately pure protein (See Part A in table, 10-20% pure) orhighly pure protein (See Part B). The results demonstrate unequivocallythat xenogenic implants of collagenous bovine bone matrix treated asdisclosed herein induces successful endochondral bone formation.

                  TABLE 1                                                         ______________________________________                                        Osteogenic Activity in rat of HF- and DCM-treated                             bovine bone matrix, rat matrix, and untreated bovine                          matrix (25 mg matarix material per implant):                                         Rat     Bovine                                                                (untreated)                                                                           (untreated) HF       DCM                                       ______________________________________                                        A. (10-20% pure OP):                                                          μg OP                                                                      1.8      +         -           -      -                                       3.8      ++        -           +      +                                       7.5      +++       +/-         ++     +++                                     B. (Purified OP:)                                                             ng OP                                                                         250      ++        -           +/     +                                       500      +++       -           +      ++                                      1000     +++       +/           ++    +++                                     ______________________________________                                         Histology score:                                                              - no bone formation                                                           + slight bone formation                                                       ++ moderate bone formation                                                    +++ extensive bone formation                                             

FIG. 3 shows the effect of HF treatment on bone formation in xenogenicrat implants, as measured by specific activity of alkaline phosphatase.It is evident from these results that osteogenic devices using an HFtreated xenogenic bone matrix induce bone, whereas devices using anuntreated matrix do not.

FIG. 4 illustrates the osteoinductive effect of water washed matrixtreated with nanogram quantities of purified OP, as indicated byspecific activity of alkaline phosphatase, for allogenic rat matrix andxenogenic bovine matrix untreated, treated with DCM alone, 99.9% DCMplus 0.1% TFA, and 90% DCM plus 10% TFA. As illustrated, DCM with lowacidified concentrations of acid enhances bone formation.

The foregoing treatment protocols generically serve to remove extraneoussoluble proteins from the bone collagen and to increase itsintraparticle surface area. While both these aspects may be important toproduction of an optimal matrix, the utility of the material of theinvention in its use as a osteogenic implant is believed to be dependentin part on increases in intraparticle surface area or porosity. Thebasis for this conclusion is apparent from a review and comparison ofFIGS. 1A through 2E. Untreated rat matrix, shown in FIGS. 1A and 2A, isactive in rats and has an obvious, open pore, high surface areastructure. The untreated bovine matrix of FIG. 1B and 2B has a lowersurface area and is inactive in rats. However, treatment of the bovinecollagen with HF (FIGS. 1C and 2C) or with DCM (FIG. 1D and 2D) producesan open pore, high surface area structure which is active xenogenically.FIGS. 1E and 2E show the appearance of bovine matrix particles whentreated with DCM but omitting the washing step. As illustrated, omissionof the wash produces a low surface area structure similar to untreatedbovine collagen, and results in an inactive matrix material. FIG. 1Fshows the structure of monkey bone collagen after treatment with HF asdisclosed above. The bone particles may be used xenogenically to inducebone. Demineralized, guanidine extracted monkey bone reportedly isineffective as a osteogenic matrix, even as an allogenic implant.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

What is claimed is:
 1. A method of manufacturing a biocompatible, invivo biodegradable matrix suitable for implantation in a mammalian host,said method comprising the steps of:A. providing insoluble,demineralized, quanidine-extracted, nonadherent bone particles,xenogenic to said host, having a mean particle diameter within the rangeof 70 mm to 850 mm; B. contacting the insoluble particles with aswelling agent to increase the intraparticle surface area andintraparticle porosity of said insoluble particles while maintaining theparticles intact; C. washing the insoluble particles which result fromstep B to remove dissociated non-collagenous components thereof; and D.close-packing the resulting porous particles to form said matrix.
 2. Themethod of claim 1 wherein the swelling agent used in step B is hydrogenfluoride.
 3. The method of claim 1 wherein the swelling agent used instep B is trifluoracetic acid.
 4. The method of claim 1 wherein theswelling agent used in step B is acetonitrile.
 5. The method of claim 1wherein the swelling agent used in step B is isopropanol.
 6. The methodof claim 1 wherein the swelling agent used in step B is acetonitrile,isopropanol, dichloromethane mixed with 0.1%-10% trifluoroacetic acid.7. The method of claim 1 wherein the swelling agent used in step B isdichloromethane.
 8. The method of claim 1 wherein the particles arewashed with a saline buffer in step C.
 9. The method of claim 1 whereinthe particles are washed with a urea-containing buffer and water in stepC.
 10. The method of claim 1 comprising the additional step of adsorbingosteogenic protein onto said particles prior to step D.
 11. The methodof claim 10 wherein the osteogenic protein is adsorbed onto saidparticles by precipitation in cold ethanol.
 12. The method of claim 10wherein the osteogenic protein is adsorbed onto said particles byincubation in a solution comprising acetonitrile and trifluoroaceticacid, followed by lyophilization.
 13. The method of claim 10 wherein theosteogenic protein present in culture medium is adsorbed onto saidparticles by incubation, followed by lyophilization.
 14. The method ofclaim 1 wherein the particles have a mean diameter within the range of150 μm to 420 μm.